Compositions and methods for introduction of odd-chain fatty acids into poultry eggs

ABSTRACT

Disclosed are compositions and methods for producing a poultry egg that is rich in odd-chain fatty acid (OCFA), particularly pentadecanoic acid (C15:0) and heptadecanoic (C17:0) acid. Poultry feed can be mixed with a compound (e.g. biomass or oil extract) that is produced from microalgae cultured to comprise elevated levels of OCFA. The OCFA-enriched poultry feed comprises an elevated level of OCFA and it can be fed to poultry that are laying eggs. As the OCFA-enriched feed is incorporated by the poultry, the resulting eggs comprise yolks that are rich in OCFA. The OCFA rich eggs can be consumed by humans as a dietary source of OCFA, to improve health.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to and is a Continuation of U.S.patent application Ser. No. 16/577,384, filed on Sep. 20, 2019, whichclaims the benefit of U.S. Provisional Application No. 62/734,437, filedon Sep. 21, 2018, each of which is hereby incorporated by reference inits entirety for all purposes.

FIELD OF THE INVENTION

This application relates generally to uses for microalgae biomass richin odd-chain fatty acid and, more specifically, to compositions andmethods for introduction of odd-chain fatty acids into poultry eggs.

BACKGROUND

The citric acid cycle can govern the energy metabolism in aerobicorganisms by producing 2 CO2, 3 NADH, 1 FADH2, and 1 ATP from theoxidation of acetyl-coA at every turn of the cycle. In addition, thecycle can provide precursors for biosynthesis of several amino acids,lipids, chlorophyll and other growth-related metabolites. The citricacid cycle is non-catalytic, which means that molecules used inbiosynthesis are replenished so that the cycle can keep generatingenergy. Regardless of how much acetyl CoA is fed into the citric acidcycle, the cycle is able to produce merely a limited amount of citricacid intermediates. Anaplerotic substrates can be used to produceintermediates that are used to replenish the oxidative capacity of thecitric acid cycle.

Anaplerosis refers to the process of replenishing the citric acid cycleintermediates and restoring energy balance of the cell (metabolichomeostasis). Odd-chain fatty acids (OCFAs) can be consideredanaplerotic because, along with acetate units, they can also releasepropionic acid which can enter the citric acid cycle through themethylmalonate pathway (OCFA catabolism). Typical dietary sources ofOCFA are milk and butter, but they have only trace amounts (<2% totalfatty acids, TFA) of pentadecanoic (C15:0) and heptadecanoic (C17:0)acid. Synthetically produced concentrated sources, such as tripentanoinand triheptanoin (e.g., oils containing C5:0 and C7:0), are notconsidered nutritional lipids. Further, current methods that involve theuse of Yarrowia lipolytica to produce odd-chain fatty acids utilizegenetic modification.

Odd-chain fatty acids are known to have potential health benefitsincluding, but not limited to, reduction of incidence of type 2diabetes, heart disease, and stroke as well as reducing incidence ofneuro-degenerative diseases such as Alzheimer's disease and Lou Gehrig'sdisease. However, a primary constraint with OCFAs is the lack of theircost-effective availability. Thus, a need exists for a natural andcost-effective source of OCFAs, particularly C15:0 and C17:0 (two longodd-chain fatty acids), that may be incorporated into a commonlyconsumed food product.

Currently, poultry eggs that are rich in OCFA are not available. Eggsare already well-accepted as a source of protein and energy, and eatenby most people. Creating an egg with 10 times more (or more) OCFA thanconventional eggs, using the techniques described herein, results in aproduct that is a quick, easy, readily available, and cost-effective wayto introduce the healthy OCFA to a human diet. Further, the process ofadding an OCFA rich biomass, or OCFA rich oil, to poultry feed is aneasy and cost-effective way to produce the eggs that are rich in OCFA.The methods described here also detail how the biomass or oil rich inOCFA can be produced.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key factors oressential features of the claimed subject matter, nor is it intended tobe used to limit the scope of the claimed subject matter.

Disclosed are compositions rich in odd-chain fatty acids, includingpentadecanoic (C15:0) and heptadecanoic (C17:0) fatty acids, andproducts rich in pentadecanoic and heptadecanoic fatty acids derivedfrom microalgae. In some embodiments, the total fatty acid profile ofthe composition includes a fraction comprising at least about sixtypercent (60%) odd-chain fatty acids (OCFA), and about twenty-five (25%)docosahexaenoic acid (DHA).

The microalgae and/or compositions rich in pentadecanoic andheptadecanoic acids may be used as a poultry feed or poultry feedingredient. Methods and systems for increasing the production orconcentration of pentadecanoic and heptadecanoic fatty acids frommicroalgae are also disclosed herein.

Further, techniques are disclosed for increasing the concentration ofOCFA in eggs, such as poultry eggs. For example, a biomass or oil richin OCFA can be incorporated into poultry feed and fed to the egg-layingpoultry. The OCFA in the feed fed to the poultry animals can result inan increase in the OCFA concentration in the fatty acid profile of theyolk of a resulting egg laid by the poultry animal.

Additionally, techniques are disclosed for creating poultry feed that isrich in OCFA. For example, a poultry feed can be mixed with a compoundderived from microalgae that is rich in OCFA. The resulting mixedpoultry feed that is rich in OCFA can be fed to poultry to produce eggsthat are rich in OCFA. For example, the OCFA eggs can be consumed byhumans as a source of OCFA in their diet to improve health.

In accordance with one or more embodiments of the present invention, anOCFA-enriched poultry feed having an elevated level of odd-chain fattyacid (OCFA) is disclosed. The OCFA-enriched poultry feed comprises:poultry feed; and a microalgae compound produced from culturingmicroalgae to produce microalgae with elevated levels of odd-chain fattyacid, the microalgae compound comprising one of a biomass and anextracted oil; and wherein the microalgae compound is mixed with thepoultry feed at an inclusion rate greater than one percent of aresulting OCFA-enriched poultry feed.

In accordance with one or more embodiments of the present invention, apoultry egg is disclosed. The poultry egg comprises an elevated level ofOCFA, the elevated level comprising an amount greater than 3% percent ofthe fatty acid profile of the yolk, wherein the poultry egg was laid bya poultry animal having been fed an OCFA-enriched poultry feedcomposition comprising an effective amount of an OCFA-rich microalgaecompound.

In accordance with one or more embodiments of the present invention, amethod for producing a poultry egg with an elevated amount of odd-chainfatty acid (OCFA) is disclosed. The method comprises: mixing a desiredamount of OCFA-rich microalgae compound into poultry feed resulting inOCFA-enriched feed composition, the desired amount comprising greaterthan one percent of the OCFA-enriched feed; feeding the OCFA-enrichedfeed composition to poultry that lays eggs; and retrieving an eggproduced by the poultry, the egg comprising an elevated amount of OCFAin the lipid profile of the yolk.

To the accomplishment of the foregoing and related ends, the followingdescription and annexed drawings set forth certain illustrative aspectsand implementations. These are indicative of but a few of the variousways in which one or more aspects may be employed. Other aspects,advantages and novel features of the disclosure will become apparentfrom the following detailed description when considered in conjunctionwith the annexed drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The innovative concepts described herein may take physical form incertain parts and arrangements of parts, a preferred embodiment of whichwill be described in detail in the specification and illustrated in theaccompanying drawings which form a part hereof, and wherein:

FIG. 1 is a line graph showing the fatty acid composition (in percentTFA) of various fatty acids in the eggs of the test group chickens;

FIG. 2 is a line graph showing the fatty acid composition (in percentTFA) of various fatty acids in the eggs of the control group chickens;

FIG. 3 is a line graph showing the mg of OCFA and DHA calculated foreach egg of the test group chickens based on the compositional profileand the total fat content of each egg;

FIG. 4 is a line graph showing the weight of both control group chickeneggs and test group chicken eggs over time; and

FIG. 5 is a bar graph detailing the results of a blind taste testevaluation of the OCFA-enriched poultry eggs versus the eggs of thecontrol group chickens.

DETAILED DESCRIPTION OF THE INVENTION

The claimed subject matter is now described with reference to thedrawings, wherein like reference numerals are generally used to refer tolike elements throughout. In the following description, for purposes ofexplanation, numerous specific details are set forth in order to providea thorough understanding of the claimed subject matter. It may beevident, however, that the claimed subject matter may be practicedwithout these specific details. In other instances, structures anddevices are shown in block diagram form in order to facilitatedescribing the claimed subject matter.

With reference to the drawings, like reference numerals designateidentical or corresponding parts throughout the several views. However,the inclusion of like elements in different views does not mean a givenembodiment necessarily includes such elements or that all embodiments ofthe innovative concepts described herein include such elements. Theexamples and figures are illustrative only and not meant to limit theinnovative concepts described herein, which is measured by the scope andspirit of the claims.

The term “microalgae” refers to microscopic single cell organisms suchas microalgae, cyanobacteria, algae, diatoms, dinoflagellates,freshwater organisms, marine organisms, or other similar single cellorganisms capable of growth in phototrophic, mixotrophic, orheterotrophic culture conditions. The term “biomass” means a compositionwherein substantially all of the components of the microalgae cellsproduced in the composition during culturing/growth remain present(e.g., in certain aspects of the invention at least about 90% of thecellular components, at least about 95% of the cellular components, orat least about 99% of the cellular components produced duringgrowth/culturing remain present).

In some embodiments, the microalgae biomass or extracts may be sourcedfrom the class Labyrinthulomycetes to make a composition that may beused to increase odd-chain fatty acid concentrations in poultry eggs.The class Labyrinthulomycetes includes species of Schizochytrium andAurantiochytrium.

Non-limiting examples of microalgae genus and species that can be usedin the compositions and methods of the claimed subject matter include:Aurantiochytrium sp., Aurantiochytrium acetophilum HS399, Chlorella sp.,Haematococcus sp., Galdieria sp., Isochrysis sp., Micractinium sp.,Porphyridium sp., Schizochytrium sp., Thraustochytrium sp., andOblongichytrium sp.

Taxonomic classification has been in flux for organisms in the genusSchizochytrium. Some organisms previously classified as Schizochytriumhave been reclassified as Aurantiochytrium, Thraustochytrium, orOblongichytrium. See Yokoyama et al. Taxonomic rearrangement of thegenus Schizochytrium sensu lato based on morphology, chemotaxonomiccharacteristics, and 18S rRNA gene phylogeny (Thrausochytriaceae,Labyrinthulomycetes): emendation for Schizochytrium and erection ofAurantiochytrium and Oblongichytrium gen. nov. Mycoscience (2007)48:199-211. Those of skill in the art will recognize thatSchizochytrium, Aurantiochytrium, Thraustochytrium, and Oblongichytriumappear closely related in many taxonomic classification trees formicroalgae, and strains and species may be re-classified from time totime. Thus, for references throughout the instant specification forSchizochytrium, it is recognized that microalgae strains in relatedtaxonomic classifications with similar characteristics toSchizochytrium, such as Aurantiochytrium, would reasonably be expectedto produce similar results.

In some embodiments, the microalgae may be cultured in phototrophic,mixotrophic, or heterotrophic culture conditions in an aqueous culturemedium. For embodiments where the microalgae is Aurantiochytriumacetophilum HS399, the Aurantiochytrium acetophilum HS399 is may becultured in either mixotrophic or heterotrophic culture conditions in anaqueous culture medium. The organic carbon sources suitable for growingmicroalgae mixotrophically or heterotrophically may comprise: acetate,acetic acid, ammonium linoleate, arabinose, arginine, aspartic acid,butyric acid, cellulose, citric acid, ethanol, fructose, fatty acids,galactose, glucose, glycerol, glycine, lactic acid, lactose, maleicacid, maltose, mannose, methanol, molasses, peptone, plant basedhydrolyzate, proline, propionic acid, ribose, sacchrose, partial orcomplete hydrolysates of starch, sucrose, tartaric, TCA-cycle organicacids, thin stillage, urea, industrial waste solutions, yeast extract,and combinations thereof. The organic carbon source may comprise anysingle source, combination of sources, and dilutions of single sourcesor combinations of sources. In some embodiments, the microalgae may becultured in axenic conditions. In some embodiments, the microalgae maybe cultured in non-axenic conditions.

Anaplerosis refers to the replenishment of the citric acid intermediatesthat have been extracted by the cell for biosynthesis. Anapleroticsubstrates, such as glucose, protein and odd-chain fatty acids, could beconverted into citric acid intermediates to restore an energy imbalanceof the cell. OCFAs are different from other anaplerotic substratesbecause they can undergo ketosis and cross the blood-brain barrier.Therefore, OCFAs have been associated with a decrease in metabolicdisease risk, and their intake has been proposed for the treatment andprevention of various gene and brain disorders. The presence of OCFAs indiet is scarce and typically limited to ruminant fat (e.g., butter),which contains only trace amounts (<2% total fatty acid (TFA)) ofpentadecanoic acid (C15:0) and heptadecanoic acid (17:0). Existingpharma OCFAs, such as tripentanoin and triheptanoin oils, are producedsynthetically, and are made of fatty acids that are not typicallypresent in a human diet. Alternatively, as described herein, a microbialprocess may be devised that can result in a natural algal oil comprisinglarge (>50 TFA) quantities of dietary (C15:0 and C17:0) OCFAs.

Typical anaplerotic substrates can include pyruvate (e.g., derived fromcarbohydrates), glutamine/glutamate (e.g., derived from protein) andprecursors of propionyl-CoA, such as odd-chain fatty acids. Anapleroticsubstrates can be used to restore energy balance in the mitochondria;and, there is a wide range of pathologies to which odd-chain fatty acidsmay provide benefits. As an example, in this aspect, odd-chain fattyacids have been experimentally used to treat: gene metabolic disorders,such as Glut1 deficiency, Fatty Acid Oxidation Disorder (FAOD), PyruvateCarboxylase Deficiency, Carnitine Palmitoyltransferase II Deficiency,Huntington, Phenylketonuria, Adult Polyglucosan Body Disease (APBD), andLong-Chain Fat Oxidation Disorders; neural disorders, such as Epilepsy,Alzheimer's Disease, and Autism Spectrum Disorder (ASD); and circulatorysystem disorders, such as Ventricular Hypertrophy, and stroke.

Dietary odd-chain fatty acids, pentadecanoic acid (C15:0) andheptadecanoic acid (C17:0), also known as margaric acid, may be derivedfrom ruminant fat (e.g., butter), and are thought to be likely derivedfrom bacterial activity in the rumen of dairy producing animals. TheseOCFAs can be found in very small amounts (e.g., <2% total fatty acids(TFA)) in some dairy products (e.g., milk and butter). Pentadecanoicacid (C15:0) and heptadecanoic acid (C17:0) have also been found to beproduced in the human gut, which may be triggered by dietary fiberintake, presumably supporting bacterial activity. Ref. 1. Because onlytrace amounts of odd-chain fatty acids (e.g., C15:0 and C17:0) arepresent in human diets, alternative sources (i.e. nutraceuticals,medical foods or therapeutics) can be used to significantly increase theintake of this type of nutrient.

Currently, merely limited amounts of odd-chain fatty acids (e.g., C15:0and C17:0) are readily available from known natural, dietary sources,such as ruminant fat. In one aspect, compositions can be created thatcomprise a higher concentration than current sources of odd-chain fattyacids such as pentadecanoic and heptadecanoic fatty acids. Further, inone aspect, a method can be devised for efficient and affectivegeneration of such fatty acids from a newly derived source.

Microalgae can produce a variety of fatty acids, the composition ofwhich can vary among different strains. As an example, thraustochytridscan accumulate lipids up to eighty-five (85%) of their dry weight; and,amongst the oleaginous microorganisms, they may be one of the fastestgrowing. Further, these organisms can be adapted to fermentationconditions (e.g., low shear sensitivity, high osmotolerance) for use inindustrial production of microbe-based oils. For example,Aurantiochytrium acetophilum HS399 (hereinafter, “HS399”) is athraustochytrid that can produce an oil containing palmitic acid (e.g.,45% total fatty acids (TFA)), n-6 docosapentaenoic acid (e.g., 8% TFA),and n-3 docosahexaenoic (e.g., 40% TFA) as the main fatty acids, withother fatty acids present in trace amounts. Pursuant to the requirementsof the Budapest Treaty, a live culture of the Aurantiochytriumacetophilum HS399 microalgae strain described herein was deposited onSep. 12, 2019 at National Center for Marine Algae and Microbiota (NCMA),located at 60 Bigelow Drive, East Boothbay, Me. 04544, USA and receivedaccession number 201909001.

The trace fatty acids of HS399 can include pentadecanoic acid (C15:0)and heptadecanoic acid (C17:0) (e.g., at <0.3% TFA). The trace fattyacids, including these two identified fatty acids, are typically ignoredin the lipid profile reports for these organisms. Odd-chain fatty acids,including pentadecanoic acid and heptadecanoic acid, are fatty acidsthat contain an odd number of carbon atoms in the structure.Pentadecanoic acid and heptadecanoic acid are both considered long chainfatty acids because they both contain more than 12 carbon atoms. OCFAsare typically related to bacterial activity (i.e. propionic acidbacteria), and are less likely to be present in algae or plants. Ref. 2

Aurantiochytrium acetophilum HS399 naturally contains trace amounts ofC15:0. The presence of trace amounts in Aurantiochytrium acetophilumHS399 suggests that the pathway responsible for the synthesis of OCFAmay be present in Aurantiochytrium acetophilum HS399. Because of thecomposition of their fatty acid profile, and their ability to be grownrapidly, microalgae such as Aurantiochytrium acetophilum HS399 providean attractive source of odd-chain fatty acids by generating odd-chainfatty acids in a more concentrated manner than other known naturalsources, such as milk fat (e.g., providing a more cost effective andefficient source of OCFA). As an example, a benefit of using microalgaein place of butter and other ruminant fat is the higher concentration ofOCFA found in them. In addition, as another example benefit, somemicroalgae oil lacks residues of phytol or phytanic acid that are oftenpresent in ruminant fat. Consumption of phytol or phytanic acid can leadto health concerns in some individuals.

Techniques have been devised that provide for an increased production ofnaturally occurring odd-chain fatty acids from microalgae. Thecultivated microalgae and/or isolated composition may be usedindividually as products or as an ingredient in a variety of products.As an example, microalgae such as Aurantiochytrium acetophilum HS399 canbe cultivated to produce a desirable fatty acid profile comprisingOCFAs, which may be isolated through various extraction processes. Inthis example, the isolated oil containing the OCFAs may comprise acomposition rich in OCFAs, such as pentadecanoic acid (C15:0) andheptadecanoic acid (C17:0). However, in one implementation, themicroalgae may be cultivated using an improved method that includes thepresence of a complex media, which can promote increased production ofthe OCFAs.

In one or more embodiments, propionic acid (e.g., and or one or morepropionates, such as the anion, salts, and/or esters of propionic acid)may be used as a precursor for production of OCFA. In thisimplementation, for example, it is likely that Aurantiochytriumacetophilum HS399 can incorporate propionic acid in its lipid generationpathway, resulting in the production of OCFAs.

Generally, fatty acid synthesis in microalgae consists of a lipidsynthesis pathway involving acetyl CoA, and some metabolic cycles. As anexample, acetyl-coenzyme A (CoA) is a universal carbon donor for fattyacid biosynthesis. Acetyl-CoA can be supplied be multiple paths, fromvarious origins, and then subsequently metabolized into malonyl-acylcarrier protein (ACP) (or Malonyl CoA) by sequential reactions (e.g.,utilizing acetyl-CoA carboxylase (ACCase) in carboxylation of A CoA). Inthis example, fatty acid synthesis follows, resulting in the productionof the fatty acids.

The genome of Aurantiochytrium acetophilum HS399 suggests that saturatedfats are synthetized through the Fatty Acid Synthase (FAS) pathway thatuses acetyl-coA as a building block for the fatty acid elongation. Theproduction of even chain fatty acids uses a malonyl-ACP as a substratefor elongation. As described herein, in one implementation, whenpropionic acid is present the acyl carrier protein (ACP) cleaves tomethylmalonyl instead of malonyl, resulting in the FAS producing ofodd-chain fatty acids instead of even chain fatty acids. Palmitic acid(C16:0) is typically the primary even chain fatty acid in HS399, whilethe primary OCFAs is typically pentadecaenoic (C15:0) instead ofheptadecanoic acid (17:0). In this implementation, fatty acid synthesisof palmitic acid (C16:0) undergoes 6 consecutive elongation cycles,while the (C15:0) OCFA undergoes only 5 elongation cycles before thefatty acid is liberated from the acyl carrier protein. In oneimplementation, a microbial process may be used to culture microalgae,which can result in a microalgae biomass and/or microalgae oilcomprising large (>50 TFA) quantities of dietary OCFA (C15:0 and C17:0).Such quantities of dietary OCFA (C15:0 and C17:0) are significantlyhigher than what is found in the naturally occurring microalgae.

In addition to the common and traditional use of propionic acid as anantimicrobial agent that kills algae, as described herein, techniqueshave been devised for propionic acid to be used to facilitate the growthof microalgae, and/or to increase OCFA production in the microalgae. Inone implementation, in this aspect, propionic acid (e.g., and/orpropionates) can be introduced into an algal bioprocess using afed-batch approach, while reducing the potential toxic effects on thealgae. According to one or more embodiments, microalgae such asAurantiochytrium acetophilum HS399 can be added to the culture medium.Propionic acid may then be added to the culture medium comprising theAurantiochytrium acetophilum HS399 in a batch, continuous feed, orfed-batch process, and cultured in a bioreactor with the culture medium.

In one embodiment, the Aurantiochytrium acetophilum HS399 cells werecultured in a two-stage axenic process which first feeds DE95 (whichcomprises about 95% dextrose) as a growth carbon source and then, aftera specified period of time, feeds propionate to the microalgae, whichcauses the microalgae to produce OCFA as a part of its fatty acidprofile. The “two-stage axenic process” refers to the first stagewherein propionate is not fed to the microalgae and the second stagewherein propionate is fed to the microalgae. The propionic acid can beadded at a ratio of at least 0.05 g of propionic acid per gram ofAurantiochytrium acetophilum HS399 biomass, in order to accumulateelevated amounts of OCFA. In one embodiment, 0.15 g of propionic acid isadded per gram of Aurantiochytrium acetophilum HS399 biomass, in orderto accumulate OCFA above 50% TFA. In another implementation, thepropionic acid can be added at a rate of above zero and up to about 3g/L per day. In one implementation, the propionic acid can be added at arate of above zero and up to about 3 g/L per day for three days,resulting in a total propionic acid addition of about 9 g/L. In oneembodiment, adding the propionate can comprise adding the propionate ina fed-batch approach into the culture medium. In one embodiment, addingthe propionate can comprise adjusting the propionate fed to produceOCFAs in a range of 5 and 70% TFAs. Optionally, anaplerotic oilcontaining concentrated amounts of OCFA can be extracted from theAurantiochytrium acetophilum HS399. In one embodiment, anaplerotic oilcan be produced from the cultured microalgae, wherein at least fivepercent of the total fatty acids (TFA) of the anaplerotic oil are OCFAs,and OCFAs make up at least one percent cell dry weight (CDW) of theanaplerotic oil. Although the above method is disclosed in detail, itshould be clearly understood that substantial benefit may still beobtained from using alternative culturing methods that would cause themicroalgae to produce elevated levels of OCFA as a part of its fattyacid profile such as, but not limited to, the methods discussed inInternational Application Number PCT/US2018/067104, Taberna et al.,which is incorporated herein in full by reference.

After the Aurantiochytrium acetophilum HS399 cells achieved the desiredOCFA profile, they were harvested, washed (i.e. diluted with water in aratio of 2:1 and centrifuged in order to remove dissolved material andsmall particles), and then dried with NATUROX® antioxidant thus formingthe Aurantiochytrium acetophilum HS399 biomass used in the experimentsdescribed herein. It should be clearly understood that other variationsof the Aurantiochytrium acetophilum HS399 biomass microalgae, includingvariations in the microalgae strains, microalgae growth or processingmethods, or variations in the stabilizers, may be used and may achievesimilar results.

The anaplerotic oil produced by microalgae can contain a substantialamount of DHA, which is a valuable nutraceutical. For example, DHA(docosahexaenoic acid) is a fatty acid that is commonly found in themeat of cold-water fish (e.g., tuna, salmon, cod, etc.). DHA has beenfound to early brain development in infants, and may improve the visionand cognitive function development. Further, DHA has been used fortreating type 2 diabetes, coronary artery disease (CAD), dementia,depression, and attention deficit-hyperactivity disorder (ADHD), as wellas improving vision and cognitive function in adults. Additionally, DHAcan be converted into eicosapentaenoic acid (EPA) in the body, which isused in the prevention and reversal of heart disease, stabilizing heartrhythm, asthma, cancer, painful menstrual periods, hay fever, lungdiseases, systemic lupus erythematosus (SLE), and certain kidneydiseases. Both EPA and DHA have been used in combination to treat highcholesterol, high blood pressure, psoriasis, Raynaud's syndrome,rheumatoid arthritis, bipolar disorder, certain inflammations of thedigestive system (ulcerative colitis), and to prevent migraine headachesin teenagers. In one or more embodiments, the anaplerotic oils producedby Aurantiochytrium acetophilum HS399 may contain OCFAs C15:0 and C17:0at 60% TFA and DHA at about 25% TFA.

In one implementation, the propionate fed approach can cause some growthinhibition in the microalgae, but may not result in a complete cultureloss of the microalgae batch. In this implementation, the fed-batchapproach may achieve similar cell densities and overall lipidaccumulation as a similar control batch with no propionic acid fed, withmerely a one-day difference. As one example, propionic acid may be fedinto the algal culture batch on demand (e.g., automatically fed using apH-auxostat fed batch system). As another example, propionic acid may befed into the microalgal batch along with a carbon source (e.g., glucose,glycerol or acetate) at a ratio below 0.1 of weight to weight (w/w) ofpropionic acid to carbon source (propionic acid/carbon source ratio). Inanother example, propionic acid may be fed along with the carbon sourceat a ratio below 0.05 w/w propionic acid to carbon source, to mitigateor avoid accumulation of propionate in the culture media. In oneexample, propionic acid may be fed into a culture at a culture pH higherthan 5. A low pH increases the toxicity of propionic acid making it moredifficult to balance the window between propionate incorporation andgrowth inhibition. Specific details and experiments regarding themethods for increasing odd-chain fatty acid production in microbialssuch as microalgae (e.g. Aurantiochytrium acetophilum HS399) areprovided in International Application Number PCT/US2018/067104, Tabernaet al., which is incorporated herein in full by reference.

Techniques and systems were devised to incorporate OCFAs (as well asDHA) found in the resulting microalgae biomass and/or microalgal oilinto poultry eggs. As an example, the biomass rich in OCFA, and/or theoil rich in OCFA, were processed into poultry feed and fed to layerchickens. In this example, the enriched feed can be fed to poultryanimals, such as chickens, resulting in the incorporation of OCFA in thecompositional profile of the poultry egg. In this way, for example, theenriched eggs can be a vehicle for delivering the OCFA to humanconsumers. As a result, a healthier egg can be created which can providepositive benefits to the cardiometabolic health (e.g., heart disease,diabetes, and stroke) of the human consumers, at a lower cost thantypical pharmaceutical or nutraceutical treatments.

In one aspect, a biomass of microalgae rich in OCFA can be fed topoultry animal, such as chickens, directly or as a processed feed. Inone implementation, in this aspect, the biomass can be fed directly tothe poultry, for example, as a supplement to other feed. In anotherimplementation, the biomass can be processed into a pelletized form andfed to the poultry, for example, as a supplement to other feed. Inanother implementation, the biomass can be combined with typical poultryfeed, and the combination can be processed into a pelletized form aspoultry feed.

In one implementation, the increased amount of OCFA in whole biomass canbe mixed with standard poultry food, for example, in the range of1.5-6.3% inclusion of the biomass into the feed. In this implementation,the mixed feed can then be fed to the poultry (e.g., chickens). Thepoultry can eat the food, for example, for chickens at the standard0.2-0.25 lbs. per day of total food intake. In this implementation, theOCFA can be incorporated into the developing egg, and will appear aspart of the fatty acid compositional profile of the yolk of theresulting poultry egg.

In another implementation, the whole biomass can be mixed with standardpoultry food, for example, in the range of 1.5-6.3% inclusion of thebiomass into the feed. In this implementation, the mixed feed can thenbe processed into pellets, which include the biomass rich in OCFA.Further, the pelletized mixed feed can be fed to the poultry, in asimilar manner as above.

In another implementation, oil derived from the biomass, such as algaloil, comprising the elevated concentration of OCFA, can be mixed withstandard poultry food, for example, in the range that may be the same asthat of the whole biomass (e.g. 1.5-6.3% inclusion) or may be less thanthat of the whole biomass (e.g., 0.5% to 4% inclusion of the oil intothe feed). In this implementation, the oil-feed mix can be fed to thepoultry. In another implementation, the oil-feed mix can be processedinto pellets, and fed to the poultry.

The resulting egg produced by the poultry, using the techniquesdescribed herein, may include an increase in OCFA over eggs produced bypoultry that are not subjected to the treated feed. For example, thepathway for digestion of fat by a chicken results in approximately 50 to65% of the fat intake directed to produce egg yolks. That is, forexample, whatever fat the chicken intakes can end up in a resulting eggyolk. Further, some of the OCFA eaten by poultry may not end up as OCFAin the resulting yolk. For example, some fatty acids may be metabolizedfor energy, stored, or incorporated in muscle. In one implementation,the composition impact on the egg can be in the range of about 1% to6.2% of total OCFA (C15:0 plus C17:0), of the total fatty acid profileof the egg.

In accordance with one or more embodiments of the present invention, anOCFA-enriched poultry feed was created. To prepare the OCFA enrichedfeed, a standard commercially available chicken feed (e.g. PETCLUB LayerCrumbles) was provided and used as the base feed. A 2.6% inclusion rateof OCFA rich Aurantiochytrium acetophilum HS399 biomass was combinedwith the standard conventional feed, thus creating the OCFA-enrichedpoultry feed. This was accomplished by mixing the correct inclusion rateof OCFA rich Aurantiochytrium acetophilum HS399 biomass with the basefeed evenly, and then forming the new composition into pellets for thesubject chickens to consume. Although a 2.6% inclusion rate of OCFA-richAurantiochytrium acetophilum HS399 biomass was used in the experiment,it should be clearly understood that substantial benefit would beobtained by using an alternative rate such as 1.5-6.3% of inclusion ofOCFA-rich Aurantiochytrium acetophilum HS399 biomass in the feed, whichwould likely result in about 250 mg to 1,000 mg of OCFA per egg. The %inclusion of the OCFA-rich Aurantiochytrium acetophilum HS399 biomass(or OCFA-rich Aurantiochytrium acetophilum HS399 oil) may be increasedor decreased depending upon how much OCFA is desired to be present inthe resulting egg yolk.

It should be noted that this is merely one example from an initial studyof the effectiveness of the techniques described herein. It is expectedthat the amount of OCFA can be even higher when fed to poultry over anextended period of time, for example, two to three times higher, ormore. It should be clearly understood that although OCFA richAurantiochytrium acetophilum HS399 biomass was used in this experimentto create the OCFA-enriched poultry feed, the same results would stillbe obtained if the OCFA rich oil was extracted from the OCFA richAurantiochytrium acetophilum HS399 biomass and the extracted OCFA richoil was mixed with the standard conventional feed. Of note, C15:0 andC17:0 fatty acids are the long odd-chain fatty acids identified in thisexample, as italicized and bolded below.

The OCFA rich Aurantiochytrium acetophilum HS399 biomass used for thisexperiment had an elevated amount of C:15 and C:17, in particular. Table1 below shows a comparison of the fatty acid profiles of the standardconventional feed alone versus the OCFA-enriched poultry feed. Withrespect to C15:0 and C17:0 in particular, the OCFA-enriched poultry feedcontained 0.88% TFA of C15:0 while the standard conventional feedcontained absolutely no C15:0 OCFA; furthermore, the OCFA-enrichedpoultry feed contained 0.84% TFA of C17:0 OCFA while the standardconventional feed only contained 0.12% TFA of C17:0 OCFA.

TABLE 1 Conventional Feed vs. OCFA treated feed Fatty Acid ConventionalFeed OCFA Enriched Feed C14:0 0.38% 0.22% C15:0 0.0 0.88% C16:0 24.4%25.4% C16:1 1.53% 2.22% C17:0 0.12% 0.84% C18:0 9.21% 8.78% C18:1 40.07%34.7% C18:2 18.44% 20.35% C18:3 0.56% 0.66% C20:4 0.0 0.0 C22:5n6 1.07%0.99% C22:6 (DHA) 0.83% 1.57% % TFA of Biomass 50.3 52.1% Protein % ofBiomass 38 36 Total OCFA 0.12% 1.72%

Analytical verification was used to ensure that the targeted compositionwas met for the OCFA-enriched poultry feed pellets. Pellets were chosenas the form of the OCFA-enriched poultry feed because the chickens wouldnot be able to differentiate between the OCFA-enriched poultry feedversus their typical base feed, which was also in pelletized form. Thishelped to ensure the chickens' consumption of the OCFA-enriched poultryfeed. Additionally, chickens tend to waste less feed when it is inpelletized form, again helping to ensure that they are consuming theOCFA-enriched poultry feed. The base feed for the control chickens issimply pressed into pellets with no additional components added and fedas noted in the treatment description further below.

For this study, 4-6 chickens were established as the control group. Thecontrol group was fed the standard commercially available base feed. Thecontrol group chickens were allowed to eat freely as they desired, whichwas at a rate of about 0.25 lbs/day/chicken. Another group of 4-6chickens made up the test group. The test group was fed with theOCFA-enriched poultry feed, with contained the 2.6% inclusion of OCFArich Aurantiochytrium acetophilum HS399 biomass. The test group chickenswere also allowed to eat freely as they desired, which was also at arate of about 0.25 lbs/day/chicken. Eggs from both the control group andthe test group were collected either daily or every other day and weredelivered for analysis twice per week on Mondays and Thursdays.

For preparation of the samples, egg yolks were separated from the eggwhites and only the yolks were analyzed. The eggs were mixed at 3-6 eggsper every 2-3 day period, freeze dried, and then submitted for analysis.

FIG. 1 is a line graph showing the fatty acid composition (in percentTFA) of various fatty acids in the eggs of the test group chickensthroughout the test duration. The raw data gathered during theexperiment is presented in Table 2 below.

TABLE 2 Percentage of TFA of Various Fatty Acids in Test Group EggsC22:5n6 C22:6n3 Day C15:0 C16:0 C17:0 C18:1 (DPA) (DHA) 0 0.00 24.400.35 40.07 1.07 0.83 4 0.00 25.93 0.33 36.05 0.93 0.76 7 0.88 25.40 0.8434.70 1.00 1.52 10 2.44 24.79 2.23 31.95 0.63 3.65 14 2.91 24.10 2.7431.47 0.40 4.52 16 2.86 23.30 2.93 32.10 0.37 4.92 20 2.66 24.13 2.7932.95 0.35 4.66 23 2.77 24.10 2.89 32.90 0.41 4.57 27 2.59 26.10 2.5531.99 0.35 4.34 30 2.71 24.26 2.98 32.73 0.39 4.69 34 2.76 25.25 2.8331.40 0.31 4.52 37 2.787 23.9377 2.921 30.783 0.35 4.64 41 2.822 23.7242.882 30.671 0.39 5.01 44 2.955 22.807 3.18 30.729 0.37 5.14 48 2.62724.025 2.889 31.315 0.48 5.48 51 2.59 23.49 2.90 32.13 0.36 5.02 55 2.3523.98 2.69 32.53 0.31 4.54 58 1.56 23.92 1.88 34.07 0.33 3.57 62 0.55425.268 0.888 36.898 0 2.036 65 0.35 26.091 0.575 36.973 0 1.6 69 0.37925.701 0.631 37.6228 0 1.575 72 0.788 25.371 1.088 36.781 0 2.1 76 1.36124.697 1.79 35.866 0 3.054 79 1.876 24.09 2.369 34.635 0 3.554 83 2.643321.7742 3.4425 32.1067 0.3524 4.594 86 2.419 22.422 3.33605 33.390.24875 4.0553 90 2.502 23.0734 2.865 32.5869 0.33351 3.9364 93 2.25523.9631 2.7715 34.3698 0.2832 3.9036 97 2.6483 23.458 3.0494 33.46110.3486 4.3577

For this experiment, OCFA-enriched poultry feed was utilized in twoseparate phases of the experiment. Phase I includes the initialintroduction of OCFA-enriched poultry feed to the chickens and feedinguntil a steady state is seen in the fatty acid profile of the eggs. ThenOCFA-enriched poultry feed is stopped to observe how quickly the OCFAlevels in the eggs drop. Phase II begins with re-introduction of theOCFA-enriched poultry feed to the chickens and then again observing theimpact on the fatty acid profile of the eggs.

Test chickens were started on OCFA containing feed on day 0, and variousfatty acids either rise of fall until a new level is reached. On day 50,test chickens were switched to a standard commercially available feed(i.e. not containing OCFA rich Aurantiochytrium acetophilum HS399biomass) and the fatty acid levels in the eggs started to fall; althoughthe OCFA levels never reached their original low levels. On day 66 PhaseII is initiated, as the test chickens were again switched to theOCFA-enriched poultry feed and the levels of fatty acids responded,rising to a level of 4.6% DHA, 3.4% C17:0 and 2.6% C15:0 which are inline with the averages in Phase I of the experiment.

FIG. 2 is a line graph showing the fatty acid composition (in percentTFA) of various fatty acids in the eggs of the control group chickensthroughout the test duration. The raw data gathered during theexperiment is presented in Table 3 below.

TABLE 3 Percentage of TFA of Various Fatty Acids in Control Group EggsC22:5n6 C22:6 Day C15:0 C16:0 C17:0 C18:1 C18:3 (DPA) (DHA) 0 0 24.40.35 40.07 0.56 1.07 0.83 10 0 26.063 0.32 38.421 0.49 0.753 0.688 160.00 25.97 0.00 37.61 0.70 0.574076 0.714121 20 0 28.28979 0 42.45510.3698 0.412054 0.512666 30 0 26.61415 0 39.24438 0.585817 0.5218640.693454 37 0 25.6591 0.2406 37.737 0.6327 0.5253 0.7426 44 0 25.46150.2431 38.1539 0.6219 0.5377 0.8605 65 0 25.149 0.3259 39.983 0.6041 00.8226 72 0 26.0368 0.2927 41.2339 0.4707 0 0.8095 79 0 25.251 0.298241.1259 0.5202 0.4817 0.6428 86 0 24.9952 0.23465 41.1792 0.5629 0.3610.681

As shown, there are no trends of significance. For example, DHA beginsat ˜0.8% and then drops as low as 0.5% but then ends at ˜0.7%. This isjust natural drift as a result of amount of feed intake the chickenshave, error in analysis, and chicken health.

The mg of OCFA and DHA were calculated for each egg of the test groupchickens based on the compositional profile and the total fat content ofeach egg. These results are shown graphically in FIG. 3 and the raw datafrom this experiment is shown in Table 4 below.

TABLE 4 Total mg of Certain Fatty Acids Per Egg C22:5n6 C22:6 Day C15:0C16:1 C17:0 C18:3 C20:3 n3 (DPA) (DHA) 0 0.0 115.4 26.4 42.3 0.0 80.762.6 4 0.0 186.6 27.8 52.2 218.4 77.1 63.5 7 61.1 154.1 58.3 45.8 174.969.4 105.5 10 188.1 144.4 172.4 56.5 148.8 48.3 281.3 14 205.4 121.4193.4 55.1 28.2 319.0 16 190.9 108.2 196.1 47.9 93.2 24.9 329.1 20 182.2130.5 191.0 44.2 97.0 23.7 318.6 23 175.9 125.5 183.7 42.3 91.1 25.7290.5 27 194.9 194.8 191.9 48.9 94.5 26.0 325.7 30 196.9 142.6 216.746.9 93.5 28.3 340.6 34 207.8 165.3 213.4 51.6 92.9 23.5 340.5 37 230.2161.9 241.3 59.8 95.7 29.0 383.6 41 191.0 129.0 195.0 48.0 86.7 26.5338.8 44 209.7 111.4 225.7 49.3 86.4 26.5 364.9 48 189.2 129.9 208.144.2 89.7 34.4 394.5 51 202.1 139.7 226.2 50.7 91.7 27.8 391.5 55 206.7170.8 236.0 56.6 105.3 27.6 398.3 58 126.8 156.6 152.9 54.2 127.2 26.7290.0 62 45.0 223.3 72.1 44.1 147.5 0.0 165.3 65 27.7 241.6 45.6 44.7147.9 0.0 126.8 69 32.7 242.9 54.4 47.0 176.2 0.0 135.8 72 62.2 217.585.9 40.3 161.7 0.0 165.7 76 101.1 196.2 133.0 37.9 139.3 0.0 226.9 79137.9 176.2 174.2 40.1 124.2 0.0 261.3 83 203.8 133.8 265.4 50.0 115.927.2 354.2 86 206.9 150.3 285.3 49.3 131.5 21.3 346.8 90 172.3 150.7197.3 41.0 108.4 23.0 271.1 93 165.8 160.1 203.8 40.4 113.3 21.3 293.897 194.7 166.6 224.2 45.1 102.6 22.8 285.3

As shown, the results are very similar to the TFA profiles noted in FIG.1 and corresponding Table 2. For example, the total fatty acid contentof Phase I and Phase II is again very similar at their steady statevalues. Additionally, it is again observed that some fatty acids, suchas C16:1, have an inverse relationship to the content of OCFA. However,this representation makes it easy to observe the amounts of fatty acidthat one would receive by consuming an egg laid by a chicken thatconsumed OCFA-enriched poultry feed (i.e. an OCFA-enriched poultry egg).For example, where one OCFA-enriched poultry egg yields about 420 mgOCFA, a typical two egg breakfast using two OCFA-enriched poultry eggswould give the consumer the recommended daily allowance of DHA (1 gramper day) and over 800 mg of OCFA per day, which is equivalent to gettingthe fatty acid nutrition out of almost 10 glasses of milk.

The weight of both control and test eggs were collected over time, andare represented in FIG. 4 and Table 5 below.

TABLE 5 Egg Weight Over Time Control Control Control Control Egg YolkWhite Shell Day weight (g) weight (g) weight (g) weight (g) 0 15.0025.00 4 7 10 14 16 52.8 17.57 29.50 5.71 20 53.4 20.50 29.00 30 55.918.30 31.20 6.40 37 55.9 16.80 33.40 5.70 44 54.7 17.30 30.50 7.00 5161.8 18.20 36.00 7.60 58 54.7 18.30 31.90 7.20 65 56.4 18.10 31.40 6.9072 59.7 18.20 34.00 7.50 79 58.6 16.60 34.70 7.40 86 58.1 19.90 31.906.30 93 55.7 17.50 30.70 7.50

As shown, there are no significant trends of weight gain, or weight lossover time in either the control group chicken eggs or in the test groupchicken eggs. There does not appear to be an impact on the overallweight of the egg from OCFA-enriched poultry feed consumption. There isa slight upward trend in the test group chicken egg total weight, and aneven more slight upward trend in the control group chicken egg totalweight. This trend is not related to the consumption of OCFA-enrichedpoultry feed, as both control group chicken eggs and test group chickeneggs have this slight trend. This non-significant trend can beattributed to the maturing of the chickens themselves. The chickens usedin the study were younger chickens, and as they matured the eggs theylaid gained weight. The control group chickens were older than the testgroup chickens by about 6 months. A typical egg is 55-62 g, and both thetest group chicken eggs and control group chicken eggs were trending inthis slightly upward direction. There was also no observable effect fromOCFA-enriched poultry feed consumption on the chicken appearance orhealth.

As shown in Tables 2-5 above and corresponding FIGS. 1-4, the amount ofOCFA collected in the yolk of the top 6 performing OCFA-enriched poultryeggs achieved a maximum average of 5.85% OCFA of the total fatty acid.This averages to about 445 mg OCFA per OCFA-enriched poultry egg. Bycomparison, this is equivalent to the amount of OCFA that a consumerwould receive by drinking 5.4 glasses of 2% milk. By further comparison,a single OCFA-enriched poultry egg would provide the equivalent intakeof OCFA to the total consumption of OCFA per capita availability per daybased on USDA data (i.e. if you take the USDA dairy intake per capita inthe US and factor in how much OCFA is in the dairy, then about 0.48 gOCFA/day is how much a consumer would receive from total dairy productsonly (dairy products include milk, cheese, yogurt, etc.). The eggs ofthe control group chickens yielded an average of about 22 mg OCFA peregg. This is 20 times less than the maximum average of the OCFA-enrichedpoultry eggs. In other words, the amount of OCFA in the OCFA-enrichedpoultry eggs was 1,922% higher than the amount of OCFA in the eggs ofthe control group chickens that were only fed standard chicken feed.

Tables 2-5 above and corresponding FIGS. 1-4 also show that the amountof OCFA that collected in the yolk of the OCFA-enriched poultry eggsduring the steady state portion of Phase I (day 16 to day 55), was 5.58%OCFA of the total fatty acid. This averages to 408 mg OCFA perOCFA-enriched poultry egg with a standard deviation of 33 mgOCFA/OCFA-enriched poultry egg. By comparison, this is equivalent to theamount of OCFA that a consumer would receive by drinking 4.8 glasses of2% milk. The eggs of the control group chickens yielded an average ofabout 22 mg OCFA per egg. This is 18.5 times less than the average ofthe OCFA-enriched poultry eggs. In other words, the amount of OCFA inthe OCFA-enriched poultry eggs was 1,755% higher than the amount of OCFAin the eggs of the control group chickens that were only fed standardchicken feed.

It is also shown in Tables 2-5 above and corresponding FIGS. 1-4 thatthe amount of OCFA that collected in the yolk of the OCFA-enrichedpoultry eggs during the steady state portion of Phase II (day 83 to day97), was 5.59% OCFA of the total fatty acid. This averages to 424 mgOCFA per OCFA-enriched poultry egg with a standard deviation of 56 mgOCFA/OCFA-enriched poultry egg. The layer chickens were very nearmolting by the end of the experiment and this may have contributed tothe higher standard deviation of the data. By comparison, this isequivalent to the amount of OCFA that a consumer would receive bydrinking 5.0 glasses of 2% milk. This value of 424 mg OCFA perOCFA-enriched poultry egg is <4% higher than that of Phase I, andconsidered to be well within the experimental error of the experiment.Therefore, Phase II may be considered a replication of the results ofPhase I. The eggs of the control group chickens yielded an average ofabout 22 mg OCFA per egg. This is 19.3 times less than the average ofthe OCFA-enriched poultry eggs. In other words, the amount of OCFA inthe OCFA-enriched poultry eggs was 1,827% higher than the amount of OCFAin the eggs of the control group chickens that were only fed standardchicken feed.

As also shown in Tables 2-5 above and corresponding FIGS. 1-4, the DHAcontent of the OCFA-enriched poultry eggs was about 340 mg DHA perOCFA-enriched poultry egg, whereas the eggs of the control groupchickens yielded only about 60 mg DHA per egg.

An in-house taste test was performed using both the control groupchicken eggs (“farm fresh” eggs), and the test group chicken eggs(OCFA-enriched poultry egg). The experiment was of interest because theDHA, which is contained in the test group chicken eggs along with theOCFA, can generate a ‘fishy’ or off taste. In particular, DHA cangenerate this off taste when it becomes oxidized, and as oxidation isexacerbated by heating, it was a question if cooking the eggs wouldcause the eggs to taste like fish, which clearly would not be a benefitfor the general appeal of the eggs to a consumer.

In a blind study, ten in-house participants evaluated both the controlgroup chicken eggs and the test group chicken eggs (containing the OCFAand DHA). Both eggs were prepared by simply cooking them “over easy” inbutter on an electric skillet with no additional salt, pepper or anyother component added. As observed in the image below, there is nodifference in appearance of the OCFA-enriched poultry eggs or farm freshegg as they are cooking on the skillet surface.

The results are shown in FIG. 5. The most important question was ofcourse whether any hint of ‘fishy’ or off taste could be detected in theOCFA-enriched poultry eggs. None of the 10 participants could find anytaste of fish or off taste in either of the eggs. The next mostimportant determination was if the OCFA or DHA in the test group chickeneggs would impact the taste such that they could be differentiated fromthe control group chicken eggs. About 70% of the participants preferredthe OCFA-enriched poultry eggs or could not tell a difference in the twoeggs. Thirty percent of the participants were able to pick out theOCFA-enriched poultry eggs, and each reported that the control eggtasted “more salty” than the OCFA-enriched poultry eggs. As they wereprepared in identical methods, this is an unexpected observation. Afinal interesting observation was that several participants thought botheggs were clearly better than anything they purchase at the conventionalshopping market.

All references, including publications, patent applications, andpatents, cited herein, are hereby incorporated by reference in theirentirety and to the same extent as if each reference were individuallyand specifically indicated to be incorporated by reference and were setforth in its entirety herein (to the maximum extent permitted by law),regardless of any separately provided incorporation of particulardocuments made elsewhere herein.

Unless otherwise stated, all exact values provided herein arerepresentative of corresponding approximate values (e.g., all exactexemplary values provided with respect to a particular factor ormeasurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate). Allprovided ranges of values are intended to include the end points of theranges, as well as values between the end points.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability, and/or enforceability of such patent documents.

The inventive concepts described herein include all modifications andequivalents of the subject matter recited in the claims and/or aspectsappended hereto as permitted by applicable law.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability, and/or enforceability of such patent documents.

The inventive concepts described herein include all modifications andequivalents of the subject matter recited in the claims and/or aspectsappended hereto as permitted by applicable law.

Although a particular feature of the disclosed techniques and systemsmay have been disclosed with respect to only one of severalimplementations, such feature may be combined with one or more otherfeatures of the other implementations as may be desired and advantageousfor any given or particular application. Also, to the extent that theterms “including”, “includes”, “having”, “has”, “with”, or variantsthereof are used in the detailed description and/or in the claims, suchterms are intended to be inclusive in a manner similar to the term“comprising.”

This written description uses examples to disclose the innovativeconcepts described herein, including the best mode, and also to enableone of ordinary skill in the art to practice the innovative conceptsdescribed herein, including making and using any devices or systems andperforming any incorporated methods. The patentable scope of theinnovative concept described herein is defined by the claims, and mayinclude other examples that occur to those skilled in the art. Suchother examples are intended to be within the scope of the claims if theyhave structural elements that are not different from the literallanguage of the claims, or if they include equivalent structuralelements with insubstantial differences from the literal language of theclaims.

In the specification and claims, reference will be made to a number ofterms that have the following meanings. The singular forms “a”, “an” and“the” include plural referents unless the context clearly dictatesotherwise. Approximating language, as used herein throughout thespecification and claims, may be applied to modify a quantitativerepresentation that could permissibly vary without resulting in a changein the basic function to which it is related. Accordingly, a valuemodified by a term such as “about” is not to be limited to the precisevalue specified. In some instances, the approximating language maycorrespond to the precision of an instrument for measuring the value.Moreover, unless specifically stated otherwise, a use of the terms“first,” “second,” etc., do not denote an order or importance, butrather the terms “first,” “second,” etc., are used to distinguish oneelement from another.

As used herein, the terms “may” and “may be” indicate a possibility ofan occurrence within a set of circumstances; a possession of a specifiedproperty, characteristic or function; and/or qualify another verb byexpressing one or more of an ability, capability, or possibilityassociated with the qualified verb. Accordingly, usage of “may” and “maybe” indicates that a modified term is apparently appropriate, capable,or suitable for an indicated capacity, function, or usage, while takinginto account that in some circumstances the modified term may sometimesnot be appropriate, capable, or suitable. For example, in somecircumstances an event or capacity can be expected, while in othercircumstances the event or capacity cannot occur—this distinction iscaptured by the terms “may” and “may be.”

The best mode for carrying out the innovative concept described hereinhas been described for purposes of illustrating the best mode known tothe applicant at the time and enable one of ordinary skill in the art topractice the innovative concepts described herein, including making andusing devices or systems and performing incorporated methods. Theexamples are illustrative only and not meant to limit the innovativeconcept described herein, as measured by the scope and merit of theclaims. The innovative concept described herein has been described withreference to preferred and alternate embodiments. Obviously,modifications and alterations will occur to others upon the reading andunderstanding of the specification. It is intended to include all suchmodifications and alterations insofar as they come within the scope ofthe appended claims or the equivalents thereof. The patentable scope ofthe innovative concept described herein is defined by the claims, andmay include other examples that occur to one of ordinary skill in theart. Such other examples are intended to be within the scope of theclaims if they have structural elements that do not differentiate fromthe literal language of the claims, or if they include equivalentstructural elements with insubstantial differences from the literallanguage of the claims.

REFERENCES

-   1. Weitkunat, K., Schumann, S., Nickel, D., Hornemann, S.,    Petzke, K. J., Schulze, M. B., . . . Klaus, S. (2017). Odd-chain    fatty acids as a biomarker for dietary fiber intake: a novel pathway    for endogenous production from propionate. The American Journal of    Clinical Nutrition, 105(6), ajcn 152702.    https://doi.org/10.3945/ajcn.117.152702.-   2. Řezanka, T., & Sigler, K. (2009). Odd-numbered very-long-chain    fatty acids from the microbial, animal and plant kingdoms. Progress    in Lipid Research, 48(3-4), 206-238.    https://doi.org/10.1016/j.plipres.2009.03.003.

What is claimed is:
 1. A method for producing a poultry egg with anelevated amount of pentadecanoic (C15:0) and heptadecanoic (C17:0)odd-chain fatty acids (OCFA), comprising: culturing thraustochytridcells to increase an amount of pentadecanoic (C15:0) and heptadecanoic(C17:0) acids in the thraustochytrid cells compared to naturallyoccurring thraustochytrid cells, resulting in OCFA-rich thraustochytridbiomass that comprises a total amount of pentadecanoic (C15:0) andheptadecanoic (C17:0) acids that is greater than 0.3% total fatty acid;mixing a desired amount of OCFA-rich thraustochytrid biomass intopoultry feed resulting in an OCFA-enriched feed composition, the desiredamount being about 1.5-6.3% of the OCFA-enriched feed composition;feeding the OCFA-enriched feed composition to poultry that lays eggs;and retrieving an egg produced by the poultry, the egg comprising anelevated amount of OCFA in the lipid profile of the yolk, wherein theelevated amount of OCFA is an amount greater than 3% of the fatty acidprofile of the yolk.
 2. The method of claim 1, wherein thethraustochytrid cells are Aurantiochytrium sp., Schizochytrium sp.,Thraustochytrium sp., or Oblongichytrium sp.
 3. The method of claim 1,wherein the step of culturing thraustochytrid cells to increase theamount of pentadecanoic (C15:0) and heptadecanoic (C17:0) acids in thethraustochytrid cells further comprises the steps of: providing aculture medium; adding thraustochytrid cells to the culture medium; andadding propionic acid to the culture medium at a rate of at least 0.05gram of propionic acid per gram of thraustochytrid cells.
 4. The methodof claim 1, wherein the OCFA-enriched feed composition comprises a totalamount of pentadecanoic (C15:0) and heptadecanoic (C17:0) acids that isabout 1.72% of the total fatty acid profile of the OCFA-enriched feedcomposition.
 5. The method of claim 1, wherein the poultry egg comprisesover 400 mg of pentadecanoic (C15:0) and heptadecanoic (C17:0) acids. 6.The method of claim 1, wherein the step of culturing thraustochytridcells to increase the amount of pentadecanoic (C15:0) and heptadecanoic(C17:0) acids in the thraustochytrid cells further comprises the stepsof: providing a culture medium; adding thraustochytrid cells to theculture medium; and adding propionic acid to the culture medium at arate of about 0.05-0.15 gram of propionic acid per gram ofthraustochytrid cells.
 7. The method of claim 1, wherein the step ofculturing thraustochytrid cells further comprises culturing thethraustochytrid cells in a two-stage axenic process, the two-stageaxenic process comprising: a first stage of adding dextrose or glucoseas a growth carbon source; and a second stage of adding propionate.
 8. Amethod for increasing an amount of pentadecanoic (C15:0) andheptadecanoic (C17:0) acids in an egg laid by a poultry animalcomprising the steps of: culturing thraustochytrid cells to increase anamount of pentadecanoic (C15:0) and heptadecanoic (C17:0) acids in thethraustochytrid cells compared to naturally occurring thraustochytridcells, resulting in OCFA-rich thraustochytrid biomass that comprises atotal amount of pentadecanoic (C15:0) and heptadecanoic (C17:0) acidsthat is greater than 0.3% total fatty acid; mixing a desired amount ofthe OCFA-rich thraustochytrid biomass into poultry feed resulting in anOCFA-enriched feed composition, the desired amount being greater than 1%of the OCFA-enriched feed composition; feeding the OCFA-enriched feedcomposition to the poultry animal that lays eggs; and retrieving an eggproduced by the poultry animal, wherein the egg comprises a total amountof pentadecanoic (C15:0) and heptadecanoic (C17:0) acids that is greaterthan 3% of the total fatty acid profile of the egg yolk.
 9. The methodof claim 8, wherein the thraustochytrid cells are Aurantiochytrium sp.,Schizochytrium sp., Thraustochytrium sp., or Oblongichytrium sp.
 10. Themethod of claim 8, wherein the step of culturing thraustochytrid cellsto increase an amount of pentadecanoic (C15:0) and heptadecanoic (C17:0)acids in the thraustochytrid cells further comprises the steps of:providing a culture medium; adding thraustochytrid cells to the culturemedium; and adding propionic acid to the culture medium.
 11. The methodof claim 8, wherein the poultry egg comprises about 250 mg to 1,000 mgof pentadecanoic (C15:0) and heptadecanoic (C17:0) acids.
 12. The methodof claim 8, wherein the OCFA-enriched feed composition comprises a totalamount of pentadecanoic (C15:0) and heptadecanoic (C17:0) acids that isabout 1.72% of the total fatty acid profile of the OCFA-enriched feedcomposition.
 13. The method of claim 8, wherein the poultry eggcomprises over 400 mg of pentadecanoic (C15:0) and heptadecanoic (C17:0)acids.
 14. The method of claim 8, wherein the step of culturingthraustochytrid cells further comprises culturing the thraustochytridcells in a two-stage axenic process, the two-stage axenic processcomprising: a first stage of adding dextrose or glucose as a growthcarbon source; and a second stage of adding propionate.
 15. A method forproducing a poultry egg with an elevated amount of pentadecanoic (C15:0)and heptadecanoic (C17:0) odd-chain fatty acids (OCFA), comprising:culturing thraustochytrid cells to increase an amount of pentadecanoic(C15:0) and heptadecanoic (C17:0) acids in the thraustochytrid cellscompared to naturally occurring thraustochytrid cells, resulting inOCFA-rich thraustochytrid biomass that comprises a total amount ofpentadecanoic (C15:0) and heptadecanoic (C17:0) acids that is greaterthan 0.3% total fatty acid; mixing a desired amount of oil, derived fromthe OCFA-rich thraustochytrid biomass, comprising elevatedconcentrations of pentadecanoic (C15:0) and heptadecanoic (C17:0) acidsinto poultry feed resulting in an OCFA-enriched feed composition, thedesired amount of oil being at least 0.5% of the OCFA-enriched feedcomposition; feeding the OCFA-enriched feed composition to poultry thatlays eggs; and retrieving an egg produced by the poultry, the eggcomprising an elevated amount of OCFA in the lipid profile of the yolk,wherein the elevated amount of OCFA is an amount greater than 3% of thefatty acid profile of the yolk.
 16. The method of claim 15, wherein thethraustochytrid cells are Aurantiochytrium sp., Schizochytrium sp.,Thraustochytrium sp., or Oblongichytrium sp.
 17. The method of claim 15,wherein the desired amount of oil is about 0.5% to 4% of theOCFA-enriched feed composition.
 18. The method of claim 15, wherein thedesired amount of oil is about 1.5% to 6.3% of the OCFA-enriched feedcomposition.
 19. The method of claim 15, wherein the step of culturingthraustochytrid cells to increase the amount of pentadecanoic (C15:0)and heptadecanoic (C17:0) acids in the thraustochytrid cells furthercomprises the steps of: providing a culture medium; addingthraustochytrid cells to the culture medium; and adding propionic acidto the culture medium at a rate of at least 0.05 gram of propionic acidper gram of thraustochytrid cells.
 20. The method of claim 15, whereinthe step of culturing thraustochytrid cells further comprises culturingthe thraustochytrid cells in a two-stage axenic process, the two-stageaxenic process comprising: a first stage of adding dextrose or glucoseas a growth carbon source; and a second stage of adding propionate.